1a.Objectives (from AD-416)
The goal of this cooperative research project is to understand the genetic complexity of field populations of the emerging species of potato cyst nematode (PCN) and to develop novel control tools to aid the eradication of PCN in Idaho. Specific objectives include:. 1)use the chorismate mutase gene as a genetic marker to evaluate genetic variations among PCN field populations,. 2)develop novel nematode resistance in potato through a plant-delivered RNAi technology.
1b.Approach (from AD-416)
The chorismate mutase gene will be cloned from different PCN field populations and the obtained gene sequences will be analyzed and compared to discover genetic variations. Transgenic potato lines expressing dsRNA targeting different parasitism genes will be generated and tested for nematode infection.
The majority of the research in 2010 was focused on propagating PCN (G. pallida) field populations and generating transgenic potato lines expressing dsRNA targeting important nematode parasitism genes. We received nematode cysts originated from eight different fields in Idaho. Due to the limited numbers of cysts received, we conducted nematode propagation on potato for each field population to obtain enough nematode cysts needed for downstream experiments. We found that potato varieties of Superior, Chieftain, and Katahdin are all suitable varieties to support G. pallida reproduction. We were able to clone three important parasitism genes GpCLE1, GpCM1, and Gp33E05 from the limited numbers of nematode cysts and conducted sequence analyses. Our results revealed that these genes share a high degree of similarity with the corresponding genes in the second species of PCN (G. rostochiensis). We further made RNAi constructs targeting each of the three parasitism genes and generated transgenic potato lines expressing each RNAi construct. Our qRT-PCR analyses confirmed dsRNA expression in most of the transgenic lines generated, revealing that the RNAi vector used is suitable for dsRNA expression in potato. These transgenic potato lines will be evaluated for nematode infection to determine if they exhibit impaired nematode parasitism. Since gene sequence conservations were observed between the two species of PCN, we are in the process of generating transgenic potato lines expressing RNAi constructs targeting gene conserved regions. These new transgenic lines will be tested for infection of both PCN species. Results from our study may suggest new methods for generating novel forms of nematode resistance in transgenic potatoes. Progress was monitored through email and phone conversations and annual reports to USDA-APHIS.